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Abstract：
The TFIIF-like Rpc53/Rpc37 heterodimer of RNA polymerase (pol) III is involved in various stages of transcription. The C-terminal region of Rpc53 dimerizes with Rpc37 to anchor on the lobe domain of the pol III cleft. However, structural and functional features of the Rpc53 N-terminal region had not been characterized previously. Here, we conducted site-directed alanine replacement mutagenesis on the Rpc53 N-terminus, generating yeast strains that exhibited a cold-sensitive growth defect and severely compromised pol III transcriptional activity. Circular dichroism and NMR spectroscopy revealed a highly disordered 57-amino acid polypeptide in the Rpc53 N-terminus. This polypeptide is a versatile protein-binding module displaying nanomolar-level binding affinities for Rpc37 and the Tfc4 subunit of the transcription initiation factor TFIIIC. Accordingly, we denote this Rpc53 N-terminus polypeptide as the TFIIIC-binding region or CBR. Alanine replacements in the CBR significantly reduced its binding affinity for Tfc4, highlighting its functional importance to cell growth and transcription in vitro. Our study reveals the functional basis for Rpc53\'s CBR in assembly of the pol III transcription initiation complex.
摘要：
RNA聚合酶（pol）III的TFIIF样Rpc53/Rpc37异二聚体参与转录的各个阶段。Rpc53的C末端区域与Rpc37二聚化，以锚定在polIII裂隙的叶域上。然而,Rpc53N端区域的结构和功能特征先前尚未表征。这里,我们在Rpc53N端进行了定点丙氨酸置换诱变，产生表现出冷敏感生长缺陷和严重损害的polIII转录活性的酵母菌株。圆二色性和NMR光谱显示Rpc53N末端高度无序的57个氨基酸的多肽。该多肽是一种通用的蛋白质结合模块，显示出对Rpc37和转录起始因子TFIIIC的Tfc4亚基的纳摩尔水平结合亲和力。因此，我们将该Rpc53N末端多肽表示为TFIIIC结合区或CBR。CBR中的丙氨酸置换显着降低了其对Tfc4的结合亲和力，突出了其对体外细胞生长和转录的功能重要性。我们的研究揭示了Rpc53的CBR在polIII转录起始复合物组装中的功能基础。
